This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The world of biomedical research needs a palette of bright, photostable, monomeric fluorescent proteins (FPs) for conventional fluorescence microscopy and for emerging FRET-based technologies. Our goal, broadly stated, is to combine directed evolution with structural analysis to engineer monomeric FPs with a range of spectral properties. A critical aspect of this work is to understand the molecular basis of chromophore biogenesis. The specific goal of this proposal is to use synchrotron radiation at APS to collect high-resolution data on a series of engineered and naturally occurring FPs. In collaboration with Steve Haddock at the Monterey Bay Aquarium Research Institute (MBARI), we discovered six new long-wavelength FPs from different color morphs of Corynactis californica. Despite a high level of sequence identity, fluorescence from these proteins spans the visible spectrum -- green, yellow, orange and red -- and includes a non-fluorescent chromoprotein and a "fluorescent timer" red FP. Structural analysis of these six will shed light on the molecular determinants of spectral variation, quantum yield and chromophore maturation. We will incorporate these observations into the design of libraries from which we will isolate monomeric variants with valuable spectral and biophysical properties. Finally, we are also collaborating with Steve Haddock at MBARI on a new reversibly photoactivatable FP, which converts from a non-fluorescent state to a brightly green fluorescent state upon excitation with the appropriate wavelength of light. Structural analysis of the "dark" and "light" states of this novel FP will shed light on the molecular details that underlie photoactivation and related phenomena (blinking, photobleaching, etc.). Ultimately, we hope to carry out time-resolved crystallographic analysis to characterize the molecular details of the dark-to-light chromophore transition.